Application of DJ-1 in treatment and diagnosis of bronchial asthma

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of DJ-1 in treatment and diagnosis of bronchial asthma. DJ-1 is used as a target in preparation or screening of drugs for bronchial asthma or allergic inflammation; a drug for treating hyperplasia of goblet cells and mucus metaplasia in bronchial asthma, and an animal model for studying inhibition of differentiation of goblet cells and mucus metaplasia. The application identifies, for the first time, an important role of a signal pathway DJ-1 / Ltc4s, which has not been found in eosinophils and lung epithelial cells before, in differentiation of secretory cells into goblet cells, and finds, for the first time, an inhibitory effect of inhibition of the DJ-1 / Ltc4s signal pathway on differentiation of goblet cells in lung epithelial cells through a large number of studies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an application of DJ-1 in treatment and diagnosis of bronchial asthma. BACKGROUND

[0002] The characteristics of bronchial asthma are airway oxidative stress, airway epithelial mucosa damage, goblet cell proliferation, chronic inflammation, and significant infiltration of inflammatory cells such as neutrophils and eosinophils. The persistence of inflammatory cells is a hallmark of the pathological features of asthma, and airway epithelial mucosa damage is the initial source of airway inflammatory response. If the airway damage cannot be repaired in time, it will lead to persistent inflammation. Therefore, in the development of respiratory diseases such as asthma, the regulation of the clearance of inflammatory cells and the repair of lung epithelial damage is crucial.

[0003] The onset of bronchial asthma is closely related to the impairment of airway mucociliary clearance. Excessive secretion of chronic mucus protein can cause ciliary movement disorders and mucus plugging in small airways, thereby causing infection, amplifying existing inflammation, and affecting gas conduction and exchange. Submucosal gland mucus / serous cells and airway epithelial goblet cells are the main sources of mucin. Goblet cells are widely distributed in the proximal to distal airways and serve as part of the airway protective barrier by secreting antibacterial proteins. Secretory (Club) cells are facultative progenitor cells that can differentiate into goblet cells. The differentiation process of lung goblet cells is strictly controlled under stable conditions. However, the underlying mechanism of goblet cell epithelialization during allergic airway inflammation is not fully understood. Currently, there are few therapeutic drugs for goblet cell epithelialization in the clinical treatment of asthma patients. Therefore, addressing airway goblet cell epithelialization is the basis for treating asthma.

[0004] DJ-1 is a Parkinson's disease-related deglycosylation gene (PARK7) located on chromosome 1p36. It has a wide range of biological functions, including antioxidant activity, transcriptional regulation, and chaperone-like functions. DJ-1 can reduce oxidative stress-induced damage to kidney and retinal epithelial cells. In a mouse model of passive cutaneous anaphylaxis, the absence of DJ-1 was shown to increase oxidative stress to promote mast cell-mediated allergic reactions. DJ-1-deficient mice showed increased lung injury and increased cell infiltration in lipopolysaccharide-induced sterile lung injury. DJ-1 has a cytoprotective effect on alveolar epithelial cells. However, it is still unclear whether DJ-1 coordinates the progression of allergic inflammation in the airway. Current treatment methods include anti-inflammatory steroids and bronchodilators to improve airflow limitation, but these methods are generally ineffective in inhibiting goblet cell epithelialization or hyperplasia. Therefore, how to inhibit airway epithelial goblet cell epithelialization or hyperplasia is the current focus of research. SUMMARY

[0005] The application aims to provide an application of DJ-1 in asthma treatment and diagnosis.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] The application includes an application of DJ-1 as a target point in preparation of a bronchial asthma diagnosis reagent; the diagnosis is performed through DJ-1 protein level, DJ-1 mRNA level or DJ-1 related signal path level in blood, bronchial alveolar lavage fluid or sputum.

[0008] The application also includes an application of DJ-1 as a target point in preparation or screening of a bronchial asthma drug.

[0009] The application also includes a drug for treating goblet cell hyperplasia or metaplasia in asthma, wherein the drug inhibits goblet cell hyperplasia or metaplasia by inhibiting DJ-1 / Ltc4s signal path in EOS.

[0010] The application also includes a drug for treating eosinophil infiltration and secretory cell proliferation in bronchial asthma, wherein the drug inhibits eosinophil infiltration and secretory cell proliferation by inhibiting DJ-1 / Ltc4s signal path in EOS.

[0011] The application also includes an animal model for studying goblet cell hyperplasia or metaplasia, which is established by inhibiting DJ-1 / Ltc4s signal path in EOS.

[0012] The application also includes an animal model for studying eosinophil infiltration and secretory cell proliferation, which is established by inhibiting DJ-1 / Ltc4s signal path in EOS. The animal model is a drug screening animal model or a drug research animal model or a treatment method research animal model or a treatment method screening animal model.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The present application first identifies the important role of a signal pathway DJ-1 / Ltc4s, which has not been found in lung epithelial cells before, in the differentiation of secretory cells into goblet cells, and first finds the inhibitory effect of the DJ-1 / Ltc4s signal pathway on the differentiation of goblet cells in lung epithelial cells through a large number of studies. The increase of DJ-1 level caused by allergic inflammation or asthma can be reduced by DJ-1 deletion, and the eosinophilia caused by allergic inflammation or asthma can be reduced by DJ-1 deletion. DJ-1 deletion leads to the decrease of Ltc4s, and the inactivation of the DJ-1 / Ltc4s signal pathway weakens the differentiation of goblet cells caused by allergic inflammation or asthma. The present application can be applied to treat eosinophil infiltration and goblet cell metaplasia or hyperplasia in allergic inflammation or asthma, and provides a new direction for precise and effective treatment of respiratory diseases such as asthma, chronic airway disease, chronic bronchitis, chronic obstructive pulmonary disease, etc. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The results of DJ-1 content in the whole lung of asthma patient sputum and chicken ovalbumin (OVA) induced allergic inflammation mice are included in the embodiments of the present application;

[0016] Figure 2 The results of the study on the abundance of eosinophils in DJ-1 knockout mice during OVA induced allergic inflammation are included in the embodiments of the present application;

[0017] Figure 3 The results of the study on the inhibition of secretory cell proliferation by the interaction between eosinophils and secretory cells are included in the embodiments of the present application;

[0018] Figure 4 The results of the study on the inhibition of goblet cell differentiation by DJ-1 knockout mice during OVA induced allergic inflammation are included in the embodiments of the present application;

[0019] Figure 5 The results of the study on the inhibition of goblet cell differentiation by the interaction between eosinophils and secretory cells in DJ-1 knockout mice are included in the embodiments of the present application;

[0020] Figure 6 The results of the study on the inhibition of leukotriene metabolism by the decrease of DJ-1 are included in the embodiments of the present application;

[0021] Figure 7 The results of the study on the promotion of secretory cell differentiation into goblet cells by leukotrienes are included in the embodiments of the present application;

[0022] Figure 8 The product of the present application is a therapeutic product, a diagnostic product and a research cell model mechanism diagram for treating respiratory diseases targeting DJ-1 (Park7). DETAILED DESCRIPTION

[0023] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with the drawings and the best embodiments.

[0024] Embodiment: DJ-1 has antioxidant activity, transcriptional regulation and chaperone-like functions, and plays an important role in various diseases. See Figure 1 , the inventors first found that the DJ-1 protein level in the sputum of asthma patients increased significantly by using ELISA technology, as shown in Figure 1 A, and the inventors first found that the DJ-1 protein level in the whole lung of the OVA-induced allergic inflammation mouse model increased significantly, as shown in Figure 1 B. The experimental results show that asthma or OVA-induced allergic inflammation can induce the increase of DJ-1 expression, which can be combined with other clinical indicators as an asthma index protein.

[0025] See Figure 2 , the DJ-1 knockout mice of C57BL / 6 / CBA background were provided by Dr. Jie Shen of Harvard Medical School. The DJ-1 gene is deleted in the whole body cells of the mice. The inventors used DJ-1 knockout mice, and injected OVA into the abdominal cavity of the mice on day 0 and day 7 for sensitization, atomized OVA from day 14 to day 18, and recovered the mice on day 19, and found that DJ-1 played an important function in the process of OVA-induced allergic inflammation. See Figure 2 A, OVA-induced allergic inflammation was used, and the bronchoalveolar lavage fluid was obtained after recovering the mice, the number of eosinophils in the bronchoalveolar lavage fluid increased, and the abundance of eosinophils decreased after the mice were knocked out of DJ-1; see Figure 2 B, after the lung tissue was enzymatically hydrolyzed into a single cell suspension, 10x single cell sequencing was performed, and various lung cells were identified and grouped, and the proportion of eosinophils in the total cells decreased after the mice were knocked out of DJ-1. The experimental results show that OVA-induced allergic inflammation in wild-type mice can cause a large number of eosinophil infiltration, and the DJ-1 knockout mice can resist eosinophil recruitment, and the DJ-1 knockout has obvious anti-inflammatory effect.

[0026] See Figure 3 , the inventors found that eosinophils interacted with secretory cells to inhibit the proliferation function of secretory cells by using the single cell sequencing results for intercellular interaction analysis, see Figure 3 A and B, 2x10 4Eosinophils were resuspended in stem cell culture medium, and 3000 secretory cells were resuspended in Matrigel. A 1:1 mixture of 100 μL and seeded in a small incubator. The mixture was then incubated at 37°C for 20 minutes to solidify. 410 μL of stem cell culture medium was added outside the incubator. The stem cell culture medium was prepared as follows: DMEM / F12 basal medium, supplemented with 10% FBS, 1% penicillin / streptomycin, 1% insulin / transferrin / selenium, 10 μM SB431542, and Y27632. This completed the establishment of the organoid co-culture model. After 8 days of culture, eosinophils reduced the colony formation rate of secretory cells; see Appendix. Figure 3 In organoid co-culture models A and C, after 8 days of culture, eosinophils reduced the size of secretory cell clones; see appendix. Figure 3 In organoid co-culture models D and E, eosinophils reduced the number of KI67+ secretory cells after 8 days of culture. The results indicate that eosinophils inhibit the proliferation of secretory cells through a contact-like inhibition mechanism.

[0027] See attached Figure 4 The inventors, using DJ-1 systemic knockout mice, discovered that systemic DJ-1 knockout in mice reduced goblet cell differentiation induced by OVA-induced allergic inflammation. (See appendix) Figure 4 In OVA-induced allergic inflammation models (A and B), systemic DJ-1 knockout in mice reduced the abundance of Alcian Blue+ cells; see appendix. Figure 4 In C and D, OVA-induced allergic inflammation models, systemic DJ-1 knockout in mice reduced the number of Clca3+ cells; see appendix. Figure 4 In a study using single-cell sequencing technology, various types of lung cells were identified and grouped. Systemic knockout of DJ-1 in mice reduced the proportion of goblet cells among airway epithelial cells. The results showed that inhibiting DJ-1 significantly suppressed goblet cell differentiation during OVA-induced allergic inflammation.

[0028] See attached Figure 5 The inventors discovered that DJ-1 deficiency weakens the role of eosinophils in promoting goblet cell differentiation. (See appendix) Figure 5 In sections A and B, the inventors discovered that eosinophils can increase the number of ClCa3+ cells in mice, thus promoting goblet cell differentiation; see appendix. Figure 5 In C and D, the inventors found that knocking out DJ-1 in eosinophils reduced the abundance of Clca3+ cells and decreased the degree of goblet cell differentiation.

[0029] See attached Figure 6 The inventors discovered that DJ-1 deficiency weakens leukotriene metabolism. (See attached document) Figure 6Fig. 1A, the inventors identified that the leukotriene metabolism in eosinophils was reduced after DJ-1 deletion by single cell sequencing technology. See attached Figure 6 Fig. 1B, the inventors identified that the expression of key enzyme genes Alox5ap, Alox5, Ltc4s in leukotriene metabolic pathway in eosinophils was reduced after DJ-1 deletion. See attached Figure 6 Fig. 1C, the inventors found that the whole lung arachidonic acid metabolism was reduced after DJ-1 deletion in mice by Bulk RNA-seq. See attached Figure 6 Fig. 1D, the inventors found that the expression of Ltc4s in whole lung was reduced. See attached Figure 6 Fig. 1E, the inventors found that the expression of Ltc4s in whole lung was reduced after DJ-1 deletion in mice by qPCR. See attached Figure 6 Fig. 1F and G, the inventors found that Cysltr1 and Cysltr2 were expressed in secretory cells by qPCR. The results showed that DJ-1 deletion reduced leukotriene metabolism in eosinophils and leukotriene receptors existed in secretory cells.

[0030] See attached Figure 7 The inventors further proved that leukotrienes could promote the differentiation of goblet cells. See attached Figure 7 Fig. 1A-C, F-H and K-M, the inventors found that leukotrienes (LTC4, LTD4, LTE4) up-regulated the levels of Clca1, Muc5ac and Spdef by qPCR detection after mRNA extraction using in vitro organoid culture model. See attached Figure 7 Fig. 1D and E, I and J, H and O, the inventors found that leukotrienes (LTC4, LTD4, LTE4) increased the abundance of Clca3+ cells by in vivo aerosol model. The results showed that leukotrienes promoted the differentiation of goblet cells.

[0031] The inventors first identified the important role of DJ-1 / Ltc4s signal pathway in eosinophil-secretory cell interaction, which was previously not found in eosinophils, activated leukotriene metabolism and promoted the differentiation of goblet cells in inflammatory airway diseases such as asthma and allergic inflammation. At the same time, eosinophils inhibited the proliferation of secretory cells. The invention can be used as a novel treatment for treating inflammation-related respiratory diseases.

[0032] The research results of the present embodiment can be applied to the preparation of products for treating goblet cell metaplasia in respiratory diseases. The products for treating goblet cell metaplasia in respiratory diseases of the present embodiment can inhibit goblet cell differentiation by inhibiting the DJ-1 / Ltc4s signal pathway.

[0033] The research results of the present embodiment can also be applied to diagnose or screen respiratory diseases, and the products can be kits, probes, primers or related markers, etc., but are not limited to the listed product types. The diagnostic or screening target of the product is DJ-1.

[0034] The research results of the present embodiment can also be applied to establish an animal model for studying the differentiation of goblet cells in respiratory diseases, and the animal model is established by activating DJ-1.

[0035] The research results of the present embodiment can also be applied to an in vitro organoid co-culture model, and the organoid co-culture model is established by activating in vitro 3D co-culture of eosinophils and lung epithelial cells.

[0036] The respiratory disease of the present embodiment can also be chronic airway disease, chronic bronchitis, asthma, chronic obstructive pulmonary disease or cystic fibrosis, but is not limited to the above-mentioned respiratory disease types.

[0037] The mechanism of the product of the present application for treating the proliferation of secretory cells and the differentiation of goblet cells in respiratory diseases is shown in the attached Figure 8 .

[0038] The above is only the preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of DJ-1 in the preparation of a diagnostic product for bronchial asthma.

2. Use according to claim 1, characterized in that, The diagnosis is made by the level of DJ-1 protein or the level of DJ-1 mRNA in blood, bronchoalveolar lavage fluid or sputum.

3. The DJ-1 / Ltc4s signaling pathway for use in establishing an animal model for studying the inhibition of goblet cell hyperplasia or metaplasia. It is established by inhibiting the DJ-1 / Ltc4s signaling pathway in EOS.

4. The use of DJ-1 / Ltc4s signaling pathway in establishing an animal model of inhibiting eosinophil infiltration and secretory cell proliferation, characterized in that, It is established by inhibiting the DJ-1 / Ltc4s signaling pathway in EOS.

5. Use according to claim 3 or 4, characterized in that, The animal model is a drug screening animal model or a drug research animal model or a treatment method research animal model or a treatment method screening animal model.

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